Functional Characterization of the 1,5-Benzodiazepine Clobazam and Its Major Active Metabolite N-Desmethylclobazam at Human GABAA Receptors Expressed in Xenopus laevis Oocytes

Abstract

The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α1,2,3,5β2γ2S GABAARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABAAR than at the α1,2,3,5β2γ2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation exerted by clobazam, N-desmethylclobazam and clonazepam at the α1β2γ2S, α2β2γ2S, α3β2γ2S and α5β2γ2S GABAARs indicate that the observed clinical differences between clobazam and 1,4-benzodiazepines are likely to arise from factors other than their respective pharmacological properties at the GABAARs as investigated here.

Using Xenopus to understand human disease and developmental disorders

“…The use of Xenopus eggs and embryos for in vivo analyses of disease gene expression and function has exploded in recent years. This model has supported important advances in our understanding of: neurological disorders including autism, Alzheimer's disease, and depression Park et al, 2015;Ramirez-Vizcarrando, Hasan, Gu, Khakhalin, & Aizenman, 2015;Ullah, Demuro, Parker, & Pearson, 2015); cancers (Green, Kwon, & Christian, 2016;Haynes-Gilmore et al, 2015;Van Nieuwenhuysen et al, 2015;Wei et al, 2016); congenital heart defects (Endicott, Basu, Khokha, & Brueckner, 2015;Silva et al, 2016;Torres-Prioris, Smith, Mohun, Fern andez, & Dur an, 2015); craniofacial and auditory malformations (Dickinson, 2016;Griffin, Sondalle, Del Viso, Baserga, & Khokha, 2015;Moody, Neilson, Kenyon, Alfandari, & Pignoni, 2015;Ramírez-Gordillo et al, 2015); diabetes (Kofent & Spagnoli, 2016;Pearl, Jarikji, & Horb, 2011;Salanga & Horb, 2015); kidney disease (Desgrange & Cereghini, 2015;Lienkamp, 2016;Stiburkova, Stekrova, Nakamura, & Ichida, 2015); Lennox-Gastaut syndrome (Hammer, Ebert, Jensen, & Jensen, 2015); and Zimmermann-Laband syndrome (Kort€ um et al, 2015).…”

Potentiating α2 subunit containing perisomatic GABAA receptors protects against seizures in a mouse model of Dravet syndrome

“…Several PAMs selective for the α 2 /α 3 subunits have been developed including L-838,417 (α 1 -sparing PAM) (McKernan et al 2000;Knabl et al 2008), TCS1105 (Taliani et al 2009), AZD7325 (Alhambra et al 2011;Zhou et al 2012) and PF-06372865 Nickolls et al 2018), although none of these is currently in clinical use as an AED. The commonly prescribed AED and anxiolytic medication CLB is a 1,5-BDZ that is known to be metabolized in vivo to active N-desmethyl clobazam (N-CLB) which has modest preference for α 2 /α 3 containing receptors (Ralvenius et al 2016; but also see Jensen et al 2014;Hammer et al 2015).…”

Systematic review of clobazam use in patients with status epilepticus

“…In an in vitro study characterizing the activity of CLB on various GABA A receptor subtypes, CLB and its metabolite nor‐clobazam (nor‐CLB) have been found to have similar activities on GABA receptor subtypes compared to clonazepam, an example of the classic 1,4 BDZ. On the other hand, CLB and its metabolite had higher efficacy on potentiating the signaling of the α 6 β 2 δ receptor, an example of the extrasynaptic GABA A receptors, compared to clonazepam . Those receptor subtypes are believed to remain intact in the setting of SE and could be a potential target for RSE management .…”

“…On the other hand, CLB and its metabolite had higher efficacy on potentiating the signaling of the a 6 b 2 d receptor, an example of the extrasynaptic GABA A receptors, compared to clonazepam. 21 Those receptor subtypes are believed to remain intact in the setting of SE and could be a potential target for RSE management. 22 The clinical significance of this differential pharmacology is not known but it could potentially contribute, at least in part, to the efficacy of CLB in causing remission in a select group of patients with SE.…”